Evaluation of structurally different benzimidazoles as priming agents, plant defence activators and growth enhancers in wheat
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Priming is a valuable, facile and well-established technique used to enhance seed quality to achieve rapid germination, establishment of stress resistance and improvement of crop yields. Different natural and synthetic priming agents have been used for better crop performance and abiotic stress management. In this study, four different benzimidazoles were selected as priming agents and their comparative effects were evaluated on different biochemical attributes including total soluble protein, total oxidant status, MDA contents, antioxidant enzymes (SOD, POD) and hydrolytic enzymes (protease, estrases) compared to control. Treatments with 2-thio-1-H-benzimidazole reduced total soluble proteins and increased total oxidant status significantly but no considerable effect was observed on other parameters. Priming with 2-(4-chlorophenyl)-1-H-benzimidazole considerably increased the total oxidant status and a little improvement was observed in total soluble proteins. Seeds primed with 1-H-benzimidazole showed a noticeable decrease in the protease activity while all other priming treatments were unable to induce any detectable change compared to control. The treatment with 2-(4-methoxyphenyl)-1-H-benzimidazole induced maximum reduction in MDA contents and POD activity. Moreover, all benzimidazole priming treatments reduced mean germination time, increased germination percentage and germination rate of wheat seeds.
KeywordsSeed priming Antioxidants Benzimidazole Hydrolytic enzymes Wheat
reactive oxygen species
mean germination time
Due to rising global population, it has been estimated that the demand for wheat is going to be doubled in 2050 . To satisfy these rising wheat demands, farmers are supposed to boost crop yields by adopting new farming strategies. In this context, enhanced seed qualities has become priority requirements to achieve uniform and rapid seedling emergence for better crop performance and finally increased yield . Seed quality is enhanced by employing facile, easily practicable and well established treatment called priming . As a result of priming treatments, germination rate increases with the development of high level stress tolerance which enhances crop yields . In fact, priming induces pre-germinative metabolism to various level in seeds depending upon their species, physiology and morphology . These specific metabolic changes trigger ATP production, de-novo synthesis of proteins and nucleic acids, activation of antioxidant enzymes and DNA repair, accumulations of phospholipids and sterols [6, 7]. The activation of these cellular mechanisms protect genome integrity, ensure rapid germination with fast seedling emergence thus help to provide high crop yields .
Around the globe wheat is the major cereal crop fulfilling almost half of the protein requirements and feeds at least one-third world population. Often wheat crop productivity is limited by slow germination rate, reduced seedling vigor, slow growth and development rates under normal and stress conditions . Under such situations, various natural and synthetic chemicals have been used as priming agents for various crops including wheat. Chemical priming offers effective opportunities for crop stress managements as it induces significant tolerance against a range of abiotic stresses . On-farm priming of wheat seeds with ascorbic acid, salicylic acid, auxins, H2O2, polyethylene glycol, kinetin and GA3 etc. has been reported to improve aforementioned germination, seedling growth, non-enzymatic and enzymatic antioxidants related attributes leading to high grain yield .
The benzimidazole and its derivatives are exceptional structural motif of wide interest exhibiting a broad spectrum of applications across a range of scientific disciplines [11, 12, 13]. The benzimidazole nucleus with varied substituents has proved as a privileged moiety with diverse potential of clinical and biological activities including antiviral, antibacterial, anti-tumor, anti-hypertensive, anti-diabetic and anti-HIV etc. [14, 15]. Compounds incorporating benzimidazole have also been used as agrochemicals with fungicidic and plant growth regulating properties . Further, they provide protection and insulate plants against various environmental stresses . Mangnucka et al. treated rye grains with 10 ppm of carbendazim and benomyl before they were allowed to germinate for 5 days . These benzimiazole-based fungicides greatly affected the biosynthesis of resorcinol and fresh and dry biomass of seedlings under thermal and light growth conditions. Seed treatments with Ambiol®, a known benzimidazole-based antioxidant increased germination, enhanced growth and improved stress tolerance in seedlings of many species [19, 20, 21]. Tomato seed treatments with Ambiol induced positive effects on germination, growth and seedling development which were passed-on to next generation. Vital parameters like photosynthesis, leaf area, percent germination, root mass and shoot mass were considerably improved in parents as well as in progeny .
In this study four different benzimidazoles were selected as wheat seed priming agents and their effects on biochemical attributes were evaluated. The subsequent sections do explain the comparative effects of these benzimidazoles on vital biochemical and germination parameters.
Materials and methods
Seed collection and priming
For this priming study, the spring wheat (Triticumaestivum L. cv. GLAXY-2013) seeds were obtained from Wheat Section, Nuclear Institute of Agriculture and Biology (NIAB), Faisalabad, Pakistan. Wheat seed priming was achieved by soaking them in aerated solutions of four different benzimidazoles with 20 and 30 ppm concentrations for 8 h. Afterwards, they were washed and dried under shade at 26 ± 2 °C until they gained original weight. Separately, seeds were soaked in distilled water for 8 h to achieve hydro-priming. Untreated or non-primed seeds were used as control for comparison in biochemical analyses and germination studies.
Biochemical analysis and germination studies
Different biochemical parameters were analyzed in primed, hydro-primed and non-primed wheat seeds to evaluate the effects of benzimidazole priming treatments. According to well-established methods for estimation and extraction of enzymes and other biochemical parameters, hydro-primed, primed and non-primed seeds were grounded using 50 mM potassium phosphate buffer with pH 7.4. At 4 °C, the grounded material was put on centrifugation at 15,000×g for 20 min and the supernatant was used for quantification studies of different enzymes. The method described by Bradford was followed for protein estimation in seed samples . Total oxidant status was determined by following the method presented by Erel et al. . This method estimates the presence of oxidants which oxidize Fe+2 to Fe+3. The method presented by Giannopolitis and Ries was followed with little modification to determine superoxide dismutase (SOD) activities . The method initially presented by Heath and Packer and then modified by Dhindsa et al. and Zhang and Kirkham was used to determine malondialdehyde (MDA) contents [27, 28, 29]. The method of Drapeau was followed for protease activity determination . The method developed by Chance and Maehly was employed for the determination of peroxidase (POD) activities . The enzyme activities were expressed on seed weight basis. According to the methods of Van Asperen , the α-naphthyl acetate and β-naphthyl acetate were used as substrates for the determination of α-esterases and β-esterases .
The recorded data was analyzed statistically by applying descriptive statistics. The significance between means was measured using Tucky’s test at 5% probability level using XL-STAT. Values presented are mean ± SD with different alphabets differ significantly from each other.
Results and discussions
The ROS are toxic by-products of aerobic metabolism and results in oxidative stress. The oxidative stress cases destruction of biomolecules like lipid, proteins, DNA and also inactivates antioxidant enzymes . Reduction in MDA level represents low levels of oxidative stress while high levels of MDA suggest overproduction of fatal free radicals [45, 46]. It may be concluded that seed priming with 30 ppm 2-(4-methoxyphenyl)-1-H-benzimidazole reduced ROS levels and oxidative stress in wheat. Wheat seed priming with polyethylene glycol has been reported to reduce MDA contents . Recently, priming treatments with mercapto-triazoles also reduced MDA content in wheat seeds representing a reduction in oxidative stress .
In conclusion, differently substituted benzimidazoles induced different effects on each biochemical parameters. Treatments with 20 ppm 2-thio-1-H-benzimidazole reduced total soluble proteins and increased total oxidant status significantly. Priming with 30 ppm 2-(4-chlorophenyl)-1-H-benzimidazole considerably increased total oxidant status and a little improvement was observed in total soluble proteins whereas treatment with its 20 ppm did not affect esterase activity. Seeds primed with 30 ppm of 1-H-benzimidazole showed a perceptible decrease in the protease activity while all other priming treatments were unable to induce any detectable change compared to control. The treatment with 30 ppm 2-(4-methoxyphenyl)-1-H-benzimidazole induced maximum reduction in MDA contents and priming with its 20 ppm decreased POD activity. All benzimidazole priming treatments reduced mean germination time, increased germination percentage and germination rate of wheat seeds and have numerous potential to be used as germination enhances under normal and stressed conditions.
AH1 (proposed the project and explained biochemical analyses), AH2 (supervised the priming and biochemical studies), TF (overall supervision and manuscript write-up), RN (interpreted the antioxidant activities), SJ (interpreted hydrolytic enzyme studies and statistical analyses), SB (enzyme studies and proof reading), AA (performed priming studies and acquisition of data), TG (critical proof reading), MA (synthesized the selected benzimidazoles). All authors read and approved the final manuscript.
The authors thankfully acknowledged the Nuclear Institute of Agriculture and Biology (NIAB), Faisalabad, Pakistan for provision of excellent lab facilities for smooth execution of this research work.
The authors declare that they have any competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published article.
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